Bacillus anthracis can induce a bacteremic phase and shock syndrome related with a high rate of mortality. A pivotal point in the pathogenesis of inhalational anthrax is macrophage infection, activation and toxin-mediated triggering to release cytokine mediators of the shock response. Studies using other intracellular pathogens indicate that infection-induced macrophage activation is independent of toxin triggering. The working hypothesis here is that blockade at the macrophage activation and/or toxin triggering steps by novel antimicrobials and toxin inhibitors will protect against anthrax-induced shock. The objectives are to use in vitro studies of infection-induced macrophage activation and toxin triggering and an in vivo model of infection-induced priming and toxin-induced triggering of the shock response to test an array of new inhibitory agents for interference at specific steps along the continuum from initial macrophage infection through shock. New antibiotics and antibiotic potentiators will be screened for access to the intracellular compartment using macrophage cell lines (one of which--an alveolar macrophage cell line--was developed for this project) and primary macrophages. Resident, inflammatory and infection-activated macrophages will be used to test the effect of macrophage activation status on drug access. Drugs that reach or are concentrated in the intracellular compartment will be tested for their effects on infection-induced, toxin-independent phenotypic and functional changes in macrophages and triggering of the NF kappa-B-dependent transcription response that is critical to cytokine activation. Related studies will test the effects of pre-screened drugs and toxin inhibitors on the combined effects of infection-induced macrophage priming and toxin triggering. Drugs and toxin inhibitors that pass in vitro screening will be evaluated further in an animal model developed to test their effects on animal priming by toxin-negative Bacillus anthracis followed with toxin-induced triggering of the shock syndrome. The long-term objectives of this project are to develop in vitro cell systems and an animal model to test new antimicrobial agents and toxin blockers for prevention of the pivotal macrophage activation response that in part mediates the fatal shock syndrome in anthrax. The goal is to develop novel therapeutic strategies to interrupt early steps in anthrax pathogenesis that can be considered for clinical testing.